The recruitment of signal transduction molecules to the membrane is crucial for the efficient coupling of extracellular signals and contractile response. Signaling molecules, including protein kinases and phosphatases, need to traffic to specific cellular locations for effective action on their downstream targets. The trafficking is dynamic and is difficult to follow.Preliminary data indicate that acetylcholine- and ceramide-induced contraction of isolated circular smooth muscle cells from the rabbit colon, is associated with: (1) PKCalpha and RhoA translocation to the membrane; (2) an immuno-complexing of PKCalpha with RhoA in the particulate fraction; and (3) an increase in the association of translocated PKCalpha and of translocated RhoA with HSP27 in the particulate fraction. Preliminary results also indicate a role for phosphorylated HSP27 in modulating the association of PKCalpha with RhoA in the particulate fraction. We have generated mutants in Human HSP27 cDNA where in, Ser-15, Ser-78, and Ser-82 were replaced with aspartate or glycine to mimic constitutively phosphorylated (3D constructs) or non-phosphorylated (3G constructs) HSP27. Preliminary observations suggest that PKCalpha and RhoA failed to translocate to the cell membrane during agonist-induced contraction in rabbit colon smooth muscle cells that were transfected with 3G constructs. Further, failure of translocation was correlated with lack of association of PKCalpha with RhoA on the membrane, a significant decrease (48.4 +/- 4% P<0.005) in the association of actin with myosin and an inhibition of acetylcholine-induced contraction. Furthermore, there was an increase in the association of PKCalpha with RhoA in cells transfected with 3D constructs. Similar results were observed in smooth muscle cells obtained from the colons of transgenic mice overexpressing the phospho-mimic form (3D) of HSP27.We therefore propose to: (1) Examine the membrane-cytoskeletal reorganization and activation of PKCalpha, RhoA and HSP27 that are activated during agonist-induced contraction. (2) Examine the interaction of RhoA, PKCalpha and HSP27 using recombinant proteins. (3) Examine the effect of expression of HSP27 mutants on the association of translocated PKCalpha and RhoA with HSP27 and the formation of a dynamic complex between HSP27-Actin-PKCalpha-RhoA in transfected cells and in transgenic mice. These pathways, not clearly defined in gastrointestinal smooth muscle, are of functional physiological significance in the normal adult and are affected due to inflammation or due to the aging process